Chlorinated and ultraviolet radiation -treated reclaimed irrigation water is the source of Aeromonas found in vegetables used for human consumption.

Wastewater is increasingly being recognized as a key water resource, and reclaimed water (or treated wastewater) is used for irrigating vegetables destined for human consumption. The aim of the present study was to determine the diversity and prevalence of Aeromonas spp. both in reclaimed water used for irrigation and in the three types of vegetables irrigated with that water. Seven of the 11 (63.6%) samples of reclaimed water and all samples of vegetables were positive for the presence of Aeromonas. A total of 216 Aeromonas isolates were genotyped and corresponded to 132 different strains that after identification by sequencing the rpoD gene belonged to 10 different species. The prevalence of the species varied depending on the type of sample. In the secondary treated reclaimed water A. caviae and A. media dominated (91.4%) while A. salmonicida, A. media, A. allosaccharophila and A. popoffii represented 74.0% of the strains in the irrigation water. In vegetables, A. caviae (75.0%) was the most common species, among which a strain isolated from lettuce had the same genotype (ERIC pattern) as a strain recovered from the irrigation water. Furthermore, the same genotype of the species A. sanarellii was recovered from parsley and tomatoes demonstrating that irrigation water was the source of contamination and confirming the risk for public health.

First record of the rare species Aeromonas schubertii from mussels: phenotypic and genetic reevaluation of the species and a review of the literature.

In a study where the prevalence of Aeromonas in shellfish was analysed, three isolates of Aeromonas schubertii were identified, representing this the first report of this species from mussels. This species was originally described in 1988 from strains isolated from extra-intestinal human infections and since then has been cited in only 18 occasions. For many years, A. schubertii was the only mannitol-negative species of the genus. However, three additional mannitol-negative species (Aeromonas simiae, Aeromonas diversa and Aeromonas australiensis) have been described. This, together with the fact that A. schubertii is a rare human pathogenic species, motivated the present study to characterize its biochemical behaviour and differentiation from the other mannitol-negative species. The molecular similarity (16S rRNA, rpoD and gyrB genes) of the strains, presence of virulence genes and antimicrobial resistance were determined. All A. schubertii strains showed the same phenotypic behaviour, i.e. they use citrate, are positive for lysine decarboxylase and DL-lactate, but negative for production of mannitol, indole and acid from sucrose and could be easily differentiated from other mannitol-negative species. All strains carried the aerA and lafA virulence genes and showed susceptibility to all antibiotics tested. Seafood could be a transmission route of this bacterium to humans.

Aeromonas lusitana sp. nov., Isolated from Untreated Water and Vegetables.

During previous studies to evaluate the phylogenetic diversity of Aeromonas from untreated waters and vegetables intended for human consumption, a group of isolates formed a unique gyrB phylogenetic cluster, separated from those of all other species described so far. A subsequent extensive phenotypic characterization, DNA-DNA hybridization, 16S rRNA gene sequencing, multi-locus phylogenetic analysis of the concatenated sequence of seven housekeeping genes (gyrB, rpoD, recA, dnaJ, gyrA, dnaX, and atpD; 4705 bp), and ERIC-PCR, were performed in an attempt to ascertain the taxonomy position of these isolates. This polyphasic approach confirmed that they belonged to a novel species of the genus Aeromonas, for which the name Aeromonas lusitana sp. nov. is proposed, with strain A.11/6(T) (=DSMZ 24095(T), =CECT 7828(T)) as the type strain.

Aeromonas dhakensis pneumonia and sepsis in a neonate Risso's dolphin Grampus griseus from the Mediterranean Sea.

A neonate Risso's dolphin Grampus griseus was found stranded alive on a beach in Catalonia, Spain. Rehabilitation attempts were unsuccessful and it died 2 d later, showing pneumonia and sepsis. A pure bacterial culture was obtained from all tissues and blood and identified as Aeromonas hydrophila using the API 20NE. However, sequencing the rpoD gene showed that the strain in fact belongs to A. dhakensis, making this the first report of fatal haemorrhagic-necrotizing pneumonia and sepsis due to this species in a marine mammal. The A. dhakensis strain GMV-704 produced ß-haemolysis, possessed several virulence genes and showed sensitivity to several antimicrobials. This study provides a new potential host for A. dhakensis, and its potential virulence in dolphins and its presence in the marine environment may warrant considering this species a potential threat to marine mammals.

Aeromonas australiensis sp. nov., isolated from irrigation water.

A Gram-negative, facultatively anaerobic bacillus, designated strain 266(T), was isolated from an irrigation water system in the south-west of Western Australia. Analysis of the 16S rRNA gene sequence confirmed that strain 266(T) belonged to the genus Aeromonas, with the nearest species being Aeromonas fluvialis (99.6% similarity to the type strain, with 6 nucleotide differences) followed by Aeromonas veronii and Aeromonas allosaccharophila (both 99.5%). Analysis of gyrB and rpoD sequences suggested that strain 266(T) formed a phylogenetic line independent of other species in the genus. This was confirmed using the concatenated sequences of six housekeeping genes (gyrB, rpoD, recA, dnaJ, gyrA and dnaX) that also indicated that A. veronii and A. allosaccharophila were the nearest relatives. DNA-DNA reassociation experiments and phenotypic analysis further supported the conclusion that strain 266(T) represents a novel species, for which the name Aeromonas australiensis sp. nov. is proposed, with type strain 266(T) (=CECT 8023(T) =LMG 26707(T)). [corrected].

Aeromonas cavernicola sp. nov., isolated from fresh water of a brook in a cavern.

Aeromonas P2973 was isolated from the water of a brook in a cavern in the Czech Republic. This isolate could not be biochemically identified at the species level, considering all updated species descriptions. Subsequent extensive phenotypic characterisation, DNA-DNA hybridisation, 16S rRNA gene sequencing and a Multi-Locus Phylogenetic Analysis (MLPA) of the concatenated sequence of 7 housekeeping genes (gyrB, rpoD, recA, dnaJ, gyrA, dnaX and atpD; 4705 bp) was employed in an attempt to ascertain the taxonomy of this isolate. Based on this polyphasic approach, we describe a novel species of the genus Aeromonas, for which the name Aeromonas cavernicola sp. nov. is proposed, with strain CCM7641(T) (DSM24474(T), CECT7862(T)) as the type strain.

A Global Survey of Hypervirulent Aeromonas hydrophila (vAh) Identified vAh Strains in the Lower Mekong River Basin and Diverse Opportunistic Pathogens from Farmed Fish and Other Environmental Sources.

Hypervirulent Aeromonas hydrophila (vAh) has emerged as the etiologic agent of epidemic outbreaks of motile Aeromonas septicemia (MAS) in high-density aquaculture of farmed carp in China and catfish in the United States, which has caused millions of tons of lost fish. We conducted a global survey to better understand the evolution, geographical distribution, and phylogeny of vAh. Aeromonas isolates were isolated from fish that showed clinical symptoms of MAS, and pure cultures were screened for the ability to utilize myo-inositol as the sole carbon source. A total of 113 myo-inositol-utilizing bacterial strains were included in this study, including additional strains obtained from previously published culture collections. Based on a gyrB phylogeny, this collection included 66 A. hydrophila isolates, 48 of which were vAh. This collection also included five new vAh isolates from diseased Pangas catfish (Pangasius pangasius) and striped catfish (Pangasianodon hypophthalmus) obtained in Cambodia and Vietnam, respectively. Genome sequences were generated from representative vAh and non-vAh isolates to evaluate the potential for lateral genetic transfer of the myo-inositol catabolism pathway. Phylogenetic analyses of each of the nine genes required for myo-inositol utilization revealed the close affiliation of vAh strains regardless of geographic origin and suggested lateral genetic transfer of this catabolic pathway from an Enterobacter species. Prediction of virulence factors was conducted to determine differences between vAh and non-vAh strains in terms of virulence and secretion systems. Core genome phylogenetic analyses on vAh isolates and Aeromonas spp. disease isolates (55 in total) were conducted to evaluate the evolutionary relationships among vAh and other Aeromonas sp. isolates, which supported the clonal nature of vAh isolates. IMPORTANCE This global survey of vAh brought together scientists that study fish disease to evaluate the evolution, geographical distribution, phylogeny, and hosts of vAh and other Aeromonas sp. isolates. In addition to vAh isolates from China and the United States, four new vAh isolates were isolated from the lower Mekong River basin in Cambodia and Vietnam, indicating the significant threat of vAh to modern aquaculture and the need for improved biosecurity to prevent vAh spread.

First Record of the Rare Species Aeromonas lusitana from Rainbow Trout (Oncorhynchus mykiss, Walbaum): Comparative Analysis with the Existing Strains.

The species Aeromonas lusitana was first described in 2016 with five strains recovered from untreated water and vegetables from Portugal. Since then, no further records exist of this species. During a surveillance study on the presence of Aeromonas in fish farms in Mexico, a new strain (ESV-351) of the mentioned species isolated from a rainbow trout was recovered. It was identified because it clustered phylogenetically with the type strain of A. lusitana based on the analysis of the rpoD gene sequences. In the present study, phenotypic characteristics, antimicrobial resistance profiles, and the presence of putative virulence genes of this novel strain (ESV-351) were determined in parallel to the five isolates from the original species description. Phenotypic differential characteristics exhibited by A. lusitana ESV-351 depicted an evident similarity to the characteristics exhibited by the other evaluated strains. However, the novel strain was positive for the production of indole using conventional methods, while the rest of the strains, including the type strain, were negative for its production. Furthermore, intermediate resistance to ampicillin, amoxicillin-clavulanic acid and cephalothin was detected in both the novel and the type strain. Five different virulence-related genes were detected in the novel strain and in the previously described strains, with the type strain exhibiting the highest number of virulence-related genes. In addition to this, the genome of the novel strain (ESV-351) was sequenced and compared with the genomes from the type strain (A. lusitana CECT 7828T) and other Aeromonas spp. The genomic analysis defined Aeromonas tecta as the closest species to A. lusitana with a highly similar number of predicted proteins. The genomic size, the number of protein-encoding genes and the number of different tRNAs, among other characteristics, make it possible to propose that the ESV-351 strain could potentially have the capacity to adapt to different environments. Genome comparison of the ESV-351 strain with the type strain revealed that both possess a similar sequence of the citrate synthase gene. In addition to this finding, the chromosomal region containing the citrate synthase locus of the novel strain exhibits some similarity to the chromosomal region in the genome of the A. hydrophila type strain and other known human pathogens, such as Vibrio cholerae. This could suggest a possible virulence role for the citrate synthase gene in A. lusitana (ESV-351).

Vibrio atlanticus sp. nov. and Vibrio artabrorum sp. nov., isolated from the clams Ruditapes philippinarum and Ruditapes decussatus.

Facultatively anaerobic marine bacteria isolated from cultured clams, Ruditapes decussatus and Ruditapes philippinarum, were previously investigated using amplified fragment length polymorphism (AFLP) and 16S rRNA gene sequence analyses. The isolates formed two AFLP clusters and belonged to the genus Vibrio, more precisely to the Splendidus clade. In this study, phylogenetic analyses based on sequences of the housekeeping genes rpoA, rpoD, pyrH, atpA and recA supported their inclusion in that clade forming two well differentiated groups with respect to the rest of the species within the clade, and confirmed that they formed two groups, separated from the rest of the species of the clade. DNA-DNA hybridization demonstrated that the isolates constitute two novel species of the genus Vibrio, which can be phenotypically differentiated from their closest relatives. The names Vibrio atlanticus sp. nov. and Vibrio artabrorum sp. nov. are proposed, with Vb 11.11(T) ( = CECT 7223(T)  = LMG 24300(T)) and Vb 11.8(T) ( = CECT 7226(T)  = LMG 23865(T)) as the type strains, respectively.

Vibrio gallaecicus sp. nov. isolated from cultured clams in north-western Spain.

A group of three motile facultative anaerobic marine bacteria were isolated from cultured Manila clams (Ruditapes philippinarum) in Galicia, north-western Spain. The strains were characterized phenotypically and genotypically. Phylogenetic analysis of the 16S rRNA gene and four housekeeping genes, RNA polymerase alpha-chain (rpoA), RecA protein (recA), the alpha-subunit of bacterial ATP synthase (atpA) and the uridine monophosphate (UMP) kinase (pyrH), indicated that these strains were closely related to the Vibrio splendidus clade. The amplified fragment length polymorphism (AFLP) fingerprints, DNA-DNA hybridizations and phylogenies of the housekeeping and 16S rRNA gene sequences showed that the three strains represented a different species from all currently described vibrios. The new species could be differentiated from its nearest neighbours on the basis of several phenotypic features. The three strains are therefore a novel species within the genus Vibrio, for which the name Vibrio gallaecicus is proposed, with the type strain being VB 8.9T(=CECT 7244T=LMG 24045T).

Diversity of Vibrios associated with reared clams in Galicia (NW Spain).

The aim of the present study was to characterize and identify vibrios isolated from cultured clams in Galicia (NW Spain). A total of 759 isolates were obtained, phenotypically characterized, grouped and assigned to the genus Vibrio. Subsequently, the genomic diversity of 145 representative strains was analyzed by means of amplified fragment length polymorphism (AFLP), which revealed a high genetic diversity amongst these isolates. Only 57 out of 145 strains could be identified to the species level, and they were distributed in 13 AFLP clusters. V. cyclitrophicus, V. splendidus and V. alginolyticus were the most abundantly represented species. Eighty-eight isolates remained unidentified, 59 were distributed over 16 clusters, while 29 were unclustered. Sequencing of the 16S rRNA and two house-keeping genes (rpoA and recA) from representative strains belonging to eight unidentified clusters with the highest number of isolates confirmed their assignation to the Vibrionaceae family, and some of these probably represent new species within the genus. The present study confirmed that the phenotypic characterization of vibrios is not sufficient to identify them at the species level. A wide diversity of vibrios was found in cultured clams from all four geographic locations analyzed. In total, more than 12 Vibrio species and at least three potential new species in this genus were identified.

Development of a PCR protocol for the detection of Aeromonas salmonicida in fish by amplification of the fstA (ferric siderophore receptor) gene.

The aims of the study were to evaluate a new PCR protocol designed to detect Aeromonas salmonicida in fish tissues and to develop a non-destructive method for the diagnosis of furunculosis. A set of primers (Fer3, Fer4), flanking a fragment of the fstA gene (coding for the ferric-siderophore receptor) was designed, showing to be sensitive and specific. When compared to PCR methods previously reported, the new protocol recognized all the 69 A. salmonicida strains evaluated, with no cross-reactions with the other bacterial species analysed. Sensitivity assays were performed in fish tissues seeded with serial dilutions of pure cultures of A. salmonicida and mixed cultures of this bacterium with Vibrio anguillarum and Aeromonas hydrophila. Detection limits obtained were of 60 and 450 bacterial cells 100 mg(-1) of tissue, respectively. Mucus and blood were evaluated in order to develop a non-destructive tool to detect the pathogen. The detection limits in seeded mucus and blood samples were 2.5 x 10(2) and 1 x 10(5) bacterial cells mL(-1), respectively. When the method was used to detect A. salmonicida in asymptomatic wild salmon, four samples of mucus and six of blood were positive, corresponding to 6 out of the 31 fish examined, whereas only one of the samples resulted positive by culture methods. It is concluded that the PCR protocol evaluated is fast, specific and sensitive to detect A. salmonicida in infected and asymptomatic fish, and will be helpful for the control of the disease through the prompt detection of carriers within fish populations.

Genome-driven evaluation and redesign of PCR tools for improving the detection of virulence-associated genes in aeromonads.

Many virulence factors have been described for opportunistic pathogens within the genus Aeromonas. Polymerase Chain Reactions (PCRs) are commonly used in population studies of aeromonads to detect virulence-associated genes in order to better understand the epidemiology and emergence of Aeromonas from the environment to host, but their performances have never been thoroughly evaluated. We aimed to determine diagnostic sensitivity and specificity of PCR assays for the detection of virulence-associated genes in a collection of Aeromonas isolates representative for the genetic diversity in the genus. Thirty-nine Aeromonas strains belonging to 27 recognized species were screened by published PCR assays for virulence-associated genes (act, aerA, aexT, alt, ascFG, ascV, ast, lafA, lip, ser, stx1, stx2A). In parallel, homologues of the 12 putative virulence genes were searched from the genomes of the 39 strains. Of the 12 published PCR assays for virulence factors, the comparison of PCR results and genome analysis estimated diagnostic sensitivities ranging from 34% to 100% and diagnostic specificities ranged from 71% to 100% depending upon the gene. To improve the detection of virulence-associated genes in aeromonads, we have designed new primer pairs for aerA/act, ser, lafA, ascFG and ascV, which showed excellent diagnostic sensitivity and specificity. Altogether, the analysis of high quality genomic data, which are more and more easy to obtain, provides significant improvements in the genetic detection of virulence factors in bacterial strains.

A Culture Independent Method for the Detection of Aeromonas sp. from Water Samples.

The genus Aeromonas is present in a wide variety of water environments and is recognised as potentially pathogenic to humans and animals. Members of this genus are often confused with Vibrio when using automated, commercial identification systems that are culture-dependent. This study describes a polymerase chain reaction (PCR) detection method for Aeromonas that is culture-independent and that targets the glycerophospholopid-cholesterol acyltransferase (gcat) gene, which is specific for this genus. The GCAT-PCR was 100% specific in artificially inoculated water samples, with a detection limit that ranged from 2.5 to 25 cfu/mL. The success at detecting this pathogen in 86 water samples using the GCAT-PCR method was identical to the conventional culturing method when a pre-enrichment step was carried out, yielding 83.7% positive samples. On the other hand, without a pre-enrichment step, only 77.9% of the samples were positive by culturing and only 15.1% with the GCAT-PCR. However, 83.7% positive samples were obtained for the GCAT-PCR when the water volume for the DNA extraction was increased from 400 µL to 4 mL. The proposed molecular method is much faster (5 or 29 h) than the culturing method (24 or 48 h) whether performed directly or after a pre-enrichment step and it will enable the fast detection of Aeromonas in water samples helping to prevent a possible transmission to humans.

Validation of a digital PCR method for quantification of DNA copy number concentrations by using a certified reference material.

Digital PCR has become the emerging technique for the sequence-specific detection and quantification of nucleic acids for various applications. During the past years, numerous reports on the development of new digital PCR methods have been published. Maturation of these developments into reliable analytical methods suitable for diagnostic or other routine testing purposes requires their validation for the intended use. Here, the results of an in-house validation of a droplet digital PCR method are presented. This method is intended for the quantification of the absolute copy number concentration of a purified linearized plasmid in solution with a nucleic acid background. It has been investigated which factors within the measurement process have a significant effect on the measurement results, and the contribution to the overall measurement uncertainty has been estimated. A comprehensive overview is provided on all the aspects that should be investigated when performing an in-house method validation of a digital PCR method.

Molecular characterization of Shewanella and Aeromonas isolates associated with spoilage of Common carp (Cyprinus carpio).

Storage in ice is a common way of preserving commercial fish species but some microorganisms can still contaminate and participate in the spoilage of the product; therefore, identification of potential harmful microbes is important. Thirteen colonies were isolated from common carp (Cyprinus carpio) that had been stored in ice, whose phenotypic identification revealed that they belonged to the genera Aeromonas (n = 5) and Shewanella (n = 8). Molecular genotyping with ERIC-PCR showed clonality only among two of the five Aeromonas isolates and for two groups (n = 3; n = 2) of the eight Shewanella isolates. Sequencing the rpoD gene showed that four Aeromonas isolates belonged to the species Aeromonas salmonicida and one to A. sobria. Of the eight Shewanella, seven isolates cluster with Shewanella putrefaciens and one with Shewanella profunda in the 16S rRNA phylogenetic tree. However, analysis of the gyrB gene showed that these eight isolates could constitute a new species closely related to S. baltica. The Shewanella and A. salmonicida isolates produce off-odours and reduce trimethylamine oxide, indicating that they might contribute to the spoilage of the fish.

Strategies to avoid wrongly labelled genomes using as example the detected wrong taxonomic affiliation for aeromonas genomes in the GenBank database.

Around 27,000 prokaryote genomes are presently deposited in the Genome database of GenBank at the National Center for Biotechnology Information (NCBI) and this number is exponentially growing. However, it is not known how many of these genomes correspond correctly to their designated taxon. The taxonomic affiliation of 44 Aeromonas genomes (only five of these are type strains) deposited at the NCBI was determined by a multilocus phylogenetic analysis (MLPA) and by pairwise average nucleotide identity (ANI). Discordant results in relation to taxa assignation were found for 14 (35.9%) of the 39 non-type strain genomes on the basis of both the MLPA and ANI results. Data presented in this study also demonstrated that if the genome of the type strain is not available, a genome of the same species correctly identified can be used as a reference for ANI calculations. Of the three ANI calculating tools compared (ANI calculator, EzGenome and JSpecies), EzGenome and JSpecies provided very similar results. However, the ANI calculator provided higher intra- and inter-species values than the other two tools (differences within the ranges 0.06-0.82% and 0.92-3.38%, respectively). Nevertheless each of these tools produced the same species classification for the studied Aeromonas genomes. To avoid possible misinterpretations with the ANI calculator, particularly when values are at the borderline of the 95% cutoff, one of the other calculation tools (EzGenome or JSpecies) should be used in combination. It is recommended that once a genome sequence is obtained the correct taxonomic affiliation is verified using ANI or a MLPA before it is submitted to the NCBI and that researchers should amend the existing taxonomic errors present in databases.

Draft genome sequences of two novel Aeromonas species recovered in association with cyanobacterial blooms.

Aeromonas aquatica and Aeromonas lacus are two new species that have been found in association with cyanobacterial blooms from recreational Finnish lakes where adverse human health effects have been recorded. Here, we present the draft genome sequences of their type strains.

Taxonomic affiliation of new genomes should be verified using average nucleotide identity and multilocus phylogenetic analysis.

The average nucleotide identity (ANI) determines if two genomes belong to the same species. Using ANI, we detected mislabeled genomes and recommend verifying with ANI and multilocus phylogenetic analysis the species affiliations of the announced genomes. The slightly different results obtained with different ANI calculation software can potentially mislead taxonomic inferences.

Re-identification of Aeromonas isolates from rainbow trout and incidence of class 1 integron and ß-lactamase genes.

Forty-eight Aeromonas isolates from rainbow trout previously identified by the 16S rDNA-RFLP technique were re-identified using 2 housekeeping genes (gyrB and rpoD). After sequencing the prevalences of the species were A. veronii (29.2%), A. bestiarum (20.8%), A. hydrophila (16.7%), A. sobria (10.4%), A. media (8.3%), A. popoffii (6.2%), A. allosaccharophila (2.1%), A. caviae (2.1%), A. salmonicida (2.1%) and one isolate (2.1%) belongs to a candidate new species "Aeromonas lusitana". Coincident identification results to the 16S rDNA-RFLP technique were only obtained for 68.8% of the isolates. PCR amplification of the enterobacterial repetitive intergenic consensus (ERIC-PCR) indicated that the 48 isolates belonged to 33 different ERIC genotypes. Several genotypes were isolated from different farms and organs in the same fish, indicating a systemic dissemination of the bacteria. The presence of genes (blaIMP, blaCphA/IMIS, blaTEM, blaSHV and intI1) that encode extended-spectrum beta-lactamases (ESBLs), metallo-beta-lactamases (MBLs) and class 1 integrons were studied by PCR. Only 39.6% (19/48) of the strains showed the presence of one or more resistance genes. The gene blaCphA/IMIS was detected in 29.2% of the isolates, followed by the intI1 (6.2%) and blaSHV (4.2%) genes. The variable region of class 1 integrons of the 3 positive isolates was sequenced revealing the presence of the gene cassette aadA1 (aminoglycoside transferase) that plays a role in streptomycin/spectinomycin resistance.